Efficacy of Plant-based Repellents Against Anopheles Mosquitoes: A Systematic Review

Mosquitoes are usually targeted using insecticides, insect growth regulators, and microbial agents. Indoor residual spraying and insecticide-treated bed nets. However, these strategies have negative effects on human health, the environment and induce resistance in a number of species. Eco-friendly tools have been recently implemented against mosquito vectors, including plant-based insecticides. To date few studies have adopted World Health Organization (WHO) Pesticide Evaluation Scheme guidelines for repellent testing against mosquitoes. This review presents a summary of recent information on development, and efficacy of plant-based repellents against Anopheles mosquitoes as well as promising new advances in the field. All eligible studies published up to April 2020 were systematically searched in several databases, namely PubMed/Medline, Scopus and Google scholar. The outcomes of interest were percentage repellency, protection time and additional properties identified in repellent compounds. A total of 27 trials met the inclusion criteria. The highest repellency effect against mosquitoes was conferred by citronella, followed by Ligusticum sinense extract, pine, Dalbergia sissoo, and Rhizophora mucronata oils with 100% protection for 8 to 14 hours. Furthermore, essential oils from plants such as lavender, camphor, catnip, geranium, jasmine, broad-leaved eucalyptus, lemongrass, lemon-scented eucalyptus, amyris, narrow-leaved eucalyptus, carotin, cedarwood, chamomile, cinnamon oil, juniper, cajeput, soya bean, rosemary, niaouli, olive, tagetes, violet, sandalwood, litsea, galbanum, and C. longa also showed >90% repellency within 8 hours against different species of Anopheles. Therefore, the review showed, essential oils and extracts of some plants could be formulated for the development of eco-friendly repellents against Anopheles species. Plant oils may serve as suitable alternatives to synthetic repellents in the future as they are relatively safe, inexpensive, and are readily available in many parts of the world.


Background
Mosquitoes serve as vectors for a wide variety of human and veterinary pathogens and parasites. Vector-borne diseases such as malaria, dengue, leishmaniasis, filariasis, and Chagas disease cause extensive morbidity and mortality, and are a major economic burden in disease-endemic countries [1,2]. Malaria, in particular, continues to have a devastating impact on infants and young children in endemic regions. In 2018 alone, over 200 million malaria cases and 0.4 million deaths we reported globally. Most of this burden was experienced by infants and young children in sub-Saharan Africa [3]. Of the 500 species of Anopheles mosquitoes thus far, approximately 50 species can transmit malaria from the bite of an infected female Anopheles spp. [1,2].
Protection from mosquito bites is one of the best approaches to reduce malaria incidences. The use of repellents to protect people from bites of mosquitoes has been acknowledged as part of an overall integrated insect-borne disease control programme [4]. Most commercial repellents are produced by using chemical components such as N, N-diethyl-metatoluamide (DEET), Allethrin, N, N-diethyl mendelic acid amide, and Dimethyl phthalate [5]. It has been reported that these chemical repellents are not safe for public health and should be used with caution. This is because of their detrimental impacts on synthetic fabric and plastic as well as toxic reactions, such as allergy, dermatitis, and cardiovascular and neurological side effects [6,7]. Prolonged and frequent use of synthetic repellents in mosquito control has resulted to development of resistance to insecticides, resurgence in mosquito populations, and adverse impact on nontarget organisms [8][9][10][11]. Accordingly, the idea of using natural mosquito repellent products as an alternative to develop new eco-friendly repellents could be an amicable solution to reverse the undesirable effects on environment and human health. In recent years, interest in plant-based repellents has been revived, as they are a rich source of bioactive phytochemicals that are safe and biodegradable into nontoxic by-products, which could be screened for insecticidal activities and mosquito repellent [12][13][14][15]. Many studies have shown that plant extracts or essential oils have repellant properties against malaria vectors around the world. Most plants produce various compounds to preventing attack from phytophagous (plant eating) insects. These chemicals fall into several categories, including repellents, feeding deterrents, toxins, and growth regulators. Based on chemical properties these compounds can be grouped into five major categories, namely nitrogen compounds (primarily alkaloids), terpenoids, phenolics, proteinase inhibitors, and growth regulators [16]. Although the primary functions of these compounds in plant defense mechanism against phytophagous insects remain unclear, many plant compounds are also effective against mosquitoes and other biting Diptera, especially volatile components released as a consequence of herbivory. The fact that several of these compounds are repellent to haematophagous insects could be an evolutionary relict from a plant-feeding ancestor, as many of these compounds evolved as repellents to phytophagous insects [17], and this repellent response to potentially toxic compounds is well conserved in the lineage of Diptera (True Flies). Plants commonly produce volatile "green leaf volatiles" when leaves are damaged in order to deter herbivores [13], and several reports have shown strong responses of mosquito odour receptors to this class of volatiles including geranyl acetate and citronellal [17], 6-methyl-5-hepten2-one and geranylacetone [18]. Furthermore, the same odour receptors that respond to DEET also respond to thujone eucalyptol and linalool in Culex quinquefasciatus [19]. In Anopheles gambiae, the DEET receptor OR83b is not only stimulated by citronellal, but it is also modulated by the TRPA1 cation channel [20]. However, it is most likely that many plant volatiles are deterrent or repellent because they have high vapour toxicity to insects [16,17]. This repellency of plant material has been exploited for thousands of years by man, most simply by hanging bruised plants in houses, a practice that is still in wide use throughout the developing countries [17]. Plants have also been used for centuries in the form of crude fumigants where plants were burnt to drive away nuisance mosquitoes and later as oil formulations applied to the skin or clothes.
A number of studies on use of repellents against malaria vectors have been conducted but it is necessary to synthesize the results of these experiments in order to inform policy decisions on use of topical repellents. Systematic reviews and meta-analysis of randomized and non-randomized studies on topical insect repellents for personal protection have been conducted but these did not expound on the efficacy and future developments of plant-based repellents which be relied on to provide a prolonged and predictable protection from species of Anopheles mosquitoes without causing side effects on human health. [5,17,21,22]. Therefore, this systematic review and meta-analysis of randomized and non-randomized controlled studies was conducted to build more understanding on the efficacy of available insect repellents and elaborate further on future development of plant-based repellents that are effective and environmental-friendly. OR "Anopheles" [Title/Abstract]). The search was limited to English publications. In addition, a manual search was conducted to identify additional pertinent articles using references from retrieved articles.

Eligibility Criteria
Studies were included in the present systematic review if they met these criteria: (a) full-text publication was written in English, (b) analyzed the repellency effects of plant extracts and essential oils against malaria vectors, Anopheles spp. mosquitoes, and, (c) reported the percentage of repellency or complete protection time. The following studies were excluded: studies exploring the repellency effect of chemical-based products, studies examining the repellency effect of animal extracts, animal models (studies not on human subjects), articles without full texts, reviews, duplicate articles, abstracts, republished data, comments, conference papers, editorials, and studies with insufficient data. In addition, studies were excluded if the information could not be extracted. A screening of titles and abstracts followed by a full-text review was performed by five investigators. All titles and abstracts were screened by five independent investigators for eligibility. If a consensus was reached, a study was excluded or selected for full-text review. If consensus was not reached, another reviewer was consulted to resolve any feasible discrepancies.

Data Extraction and Analysis
After screening for the eligible studies, the following information was extracted: name of the first author, country of origin, journal details, publication year, condition of study (field or laboratory), plant name, Anopheles species, concentration of repellents, repellency percentage and complete protection time using a standardized data collection form. All data were independently extracted by two reviewers and disagreements were solved by discussion, and if necessary, a third author was involved.

Results
A total of 285 studies were identified in the initial literature search. Of the 216 studies excluded, 102 were duplicated studies; 39 were not on repellency effect of plants on Anopheles spp., 7 were review publications; 6 investigated the repellency impact of chemical-based repellents or animal extracts; 11 studies were conducted on laboratory animals and 10 studies had not reported adequate data concerning the percentage of repellency or 100% protection time. The primary eligibility process yielded 27 documents. Literature search in the references of reviews and other databases search provided 3 additional articles. A total of 15 studies were conducted in different countries, including Brazil, India and Thailand.

Essential Oils
Essential oils distilled from members of the Lamiaceae (mint family that includes most culinary herbs), Poaceae (aromatic grasses) and Pinaceae (pine and cedar family) are commonly used as insect repellents throughout the globe as shown in Table 1. Many members of these families are used in rural communities through burning or hanging them within homes [34]. In Europe and North America there is a strong history of the use of oils that date back to Ancient times. Almost all plants used as repellents are also used for food flavouring or in the perfume industry, which may explain the association with these oils as safer natural alternatives to DEET despite many oils causing contact dermatitis [33]. Many commercial repellents contain several plant essential oils either for fragrance or as repellents including peppermint, lemongrass, geraniol, pine oil, pennyroyal, cedar oil, thyme oil and patchouli. The most effective of these include thyme oil, geraniol, peppermint oil, cedar oil, patchouli and clove that have been found to repel malaria, filarial and yellow fever vectors for a period of 60 -180 mins [44][45][46][47][48][49][50][51][52][53][54][55][56]. Most of these essential oils are highly volatile and this contributes to their poor longevity as mosquito repellents. However, this problem can be addressed by using fixatives or careful formulation to improve their longevity. For example, oils from turmeric and hairy basil with addition of 5% vanillin repelled three species of mosquitoes for a period of 6-8 hours depending on the mosquito species [34]. Although essential oils are exempt from registration through the EPA, they can irritate the skin and their repellent effect is dependent on formulation and concentration. Repellents containing only essential oils in the absence of an active ingredient such as DEET should not be recommended as repellents for use in disease endemic areas, and those containing high levels of essential oils could cause skin irritation, especially in the presence of sunlight.

Citronella
Essential oils and extracts derived from plants in the citronella genus (Poaceae) are commonly used as ingredients of plant-based mosquito repellents (Table 1), mainly Cymbopogon nardus that is sold in Europe and North America in commercial preparations. The repellency effect of citronella has been documented in several studies describing citronella as an essential oil extracted from the stems and leaves of different species of lemongrass (Cymbopogon spp.) [23]. A study on mosquitoes repellency found that citronella obtained from lemongrass has a 100% repellency effect against Anopheles culicifacies for 11 hours [24]. Recently, several studies have also reported that citronella can repel different Anopheles spp. for 8 to 6 hours [15,22,2]. Different concentration of citronella essential oil (100 µl to 0.1 ml) have also been shown to have significant repellency for 4 to 0.8 of 100% protection time against almost all mosquitoes vectors of human importance in a number of studies (field and laboratory-based) across the globe [15,17,[26][27][28]. However, the percentage repellency of citronella varies (52 to 85%) depending on the concentration of extracts and mosquito species tested. A few studies have analyzed the efficacy of essential oils extracted from citronella for repellency effect against arthropods [29]. Citronella-based repellents only protect from host-seeking mosquitoes for about two hours although formulation of the repellent is very crucial. The results indicate citronella, which contain citronellal, citronellol, geraniol, citral, a pinene, and limonene, is as effective as DEET. However, the oils from citronella are highly volatile thus causing reducing its efficacy and protection. This observation has unraveled the need to mix essential oil of Cymbopogon winterianus with a large molecules such as vanillin (5%), to prolong protection time and reduce the evaporation rate of the volatile oil [30]. Recently, the use of nanotechnology has enabled slow release of oils, thus prolonging protection time [31]. The encapsulated citronella oil nanoemulsion was initially prepared by high-pressure homogenization of 2.5% surfactant and 100% glycerol, to develop stable droplets that increase the retention of the oil and slow down release. The release rate relates well to the protection time so that a decrease in release rate can prolong mosquito protection time [31]. Another strategy adopted to prolong the effect of plant-based repellents is microencapsulation using gelatin-arabic gum microcapsules.
This approach can sustain complete citronella repellency effect for up to 30 days on treated fabric stored at room temperature [32]. The use of such advanced technologies to sustain and improve the performance of natural repellents may transform the repellent market and promote the use of plant oils as eco-friendly and long-lasting mosquito repellents. myrcene linalool a-pinene eucalypto alloparinol camphor limonene Govere et al., [39] L. javanica a -terpeneol verbenone Seyoum et al., [40,37] Kenya Tanzania  Moore et al., [35] 100% essential oil combined with vanillin 5% applied topically 100% protection against An. stephensi for 8 hours laboratory study Tawatsin et al., [45] 100% essential oil combined with vanillin 5% applied topicall 100% protection against An. dirus for 8 hour laboratory study

Current Developments in Plant-based Repellents Against Mosquitoes
The field of plant-based repellents is advancing with consumer demand for protection against arthropod bites using safe, pleasant to use and environmentally sustainable products. Perhaps the most important consideration is improving the longevity of those repellents that are effective but volatile such as citronella. Several studies looked at improving formulations of plant oils to increase their longevity through development of nanoemulsions [31], enhanced formulations and fixatives [46]; while alternate uses such as spatial activity [47] and excito-repellency [48] have also been investigated. There has been a single clinical study of PMD to lower malaria incidence [49]. This is an exciting discovery since PMD can be recovered by distilling the leaves of E. citroidora or chemical modification of citronellal [50] available from plants of the genus Cymbopogon. These plants are already commercially cropped in malaria endemic countries including South America, especially Brazil (over 5 million trees), southern China, India, Sri Lanka, Congo (Zaire), Kenya and most countries in southern Africa, where it is grown for essential oil production and timber. Local production of insect repellent would remove the high cost of importation in developing countries. New developments have also been seen in understanding the function of plant-based repellents in insects. Several studies have investigated the behavioural mode of action of repellents through structure-activity studies of contact versus spatial repellency [51], or olfactometry that demonstrated that DEET inhibited mosquito response to human odour whereas Ocimum forskolei repels but does not inhibit response to human odour [52]. Furthermore, it has been shown that citronellal directly activates cation channels, which is similar to the excitorepellent effect of pyrethrin -another plant based terpine [53], unlike DEET which has inhibitory effect [3]. The field of repellent development from plants is extremely fertile due to wealth of insecticidal compounds found in plants as defences against insects. The modern pyrethroids that are the mainstay of the current malaria elimination program that is making excellent progress, are synthetic analogues based on the chemical structure of pyrethrins, discovered in the pyrethrum daisy, Tanacetum cinerariifolium from the Dalmation region and Tanacetum coccineum of Persian origin. The field of plant-based repellent evaluation and development had become far more rigorous in recent years and developments in methods of dispensing plant-based volatiles means that extension in the duration of repellency and consequent efficacy of plant-based repellents will be possible in future.

Discussion
Widespread pest and disease vector resistance against chemical control strategies pose a huge impediment toward successful disease and pest control. To overcome this problem, development of effective approaches is paramount. The field of herbal repellents is rapidly evolving with the increased demand for mosquitoes' repellents that are safe, pleasant to usage and eco-friendly. Since high cost of developing repellants could delay development and successful deployment of pest and vector control programs, use of local florae as repellents offer an attractive alternative. Essential oils and plant extracts are emerging as potential agents for Anopheles spp. control because they are easy-to-administer, low-cost, and risk-free properties. In the present systematic review, L. sinense extract was found to have the highest repellency effect against Anopheles mosquitoes, followed by citronella, pine, D. sissoo, peppermint and R. mucronata oils. Complete protection time ranged from 9.1 to 11.5 hours. Essential oils from plants such as lavender, camphor, catnip, geranium, jasmine, broad-leaved eucalyptus, lemongrass, lemonscented eucalyptus, amyris, narrow-leaved eucalyptus, carotin, cedarwood, chamomile, cinnamon oil, juniper, cajeput, soya bean, rosemary, niaouli, olive, tagetes, violet, sandalwood, litsea, galbanum, and C. longa also shown good repellency of up to 8 hours' complete repellency against different species of Anopheles genus.
Noteworthy is that the review identified the exact mechanism of action of these plants in preventing Anopheles spp. bites is yet to be fully understood. Nevertheless, Citronella extract, one of the most investigated plant repellency effect of various mosquitoes, has been reported to have active compounds that repel mosquitoes. these include eugenol, eucalyptol, camphor, linalool, citral, and citronellal [54]. Data collected so far propose that these agents interfere with olfactory receptors of mosquitoes [55]. A recent study revealed that An. gambiae is able to detect molecules derived from citronellal using olfactory neurons in the antenna that is controlled by TRPA1 gene, activated directly by the molecule with high potency [56]. Another study found that citronellal directly activates cation channels [55], This mechanism is similar to the excite-repellent effect of pyrethrin, a plant-based terpine [17], but contrasts with the inhibitory effect of DEET [56]. Although the protection time of citronella oil is shorter than that of DEET, Citronella oil could provide sufficient protection time against mosquitoes. For other plants, the underlying mechanism remains to be elucidated.
Improving the formulations of plant extracts is key to elevate longevity of volatile plant-based repellents. This could be achieved by developing encapsulated nano emulsions, Mosquitoes: A Systematic Review improved formulations and fixatives. While alternative uses such as excite-repellency and spatial activity have already been examined [56], there is need for caution when interpreting the findings for several reasons. First, a poorly inspected confounding aspect is the effect of sweating on the effectiveness and protection time of repellents, which are water-soluble, might limit the comparison of the effect of repellents. Second, in field studies, the number of human volunteers as well as the season during which a trial is conducted differed among studies included in this review. Climate change could also affect mosquito behavior and the variance is controlled by standardizing humidity temperature in 'arm-in-cage' trials; however, these parameters are not always similar in different trials or conform to the mosquito environment standards. Third, it should be highlighted that some plant compounds are irritating to the skin and/or highly toxic to mammals, and natural does not equate to safe. Thus, plants with potential repellency properties should be tested for their possible undesired effects before adoption as alternative mosquito repellant products. Fourth, some studies have shown that formulation play a significant role in the effectiveness of a repellants [34].
A few studies have focused on the search for active compounds than on optimal formulations [34,54]. In this study, many studies showed the effectiveness of plant repellents against Anopheles spp. mosquitoes. However, when focusing on Anopheles subspecies, there were only a few publications indicating the efficacy of each plant, which resulted in a difficulty to reach a robust conclusion regarding the best herbal candidates to develop new commercial repellents. Thus more research is required. Finally, current studies are difficult to be compared and the repellency effectiveness may also differ among subspecies. Unfortunately, a few studies aimed to compare repellency efficacy of a special plant on subspecies of Anopheles. The heterogeneity in the results of previous studies might be explained by differences in compound concentrations, application dosages, mosquito species, formulations and the assessment method of repellency. For instance, some trials recorded protection time after mosquitoes landed on the subject. Most studies however, recorded time until mosquito bite occurred. Given the heterogeneity in the studies included in the current systematic review, future research assessing the repellent impacts should provide clear description of repellents, characteristics of volunteers in field trials, mosquito species, and outcome measures.

Conclusion
The results of this review show that some plants essential oils and extracts have significant repellent activity against Anopheles spp. mosquitoes. The studies conducted in the last two decades have focused on the search for new natural repellents and some plants displayed good repellent properties. However, few natural products have been developed so far. This review calls underscore the need to understand the potential role of plant-derived repellents contribute in disease control.